A thermo-responsive and self-healing liposome-in-hydrogel system as an antitubercular drug carrier for localized bone tuberculosis therapy

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Abstract

Isoniazid (INH) is a first-line therapy for bone tuberculosis (TB), but its clinic benefits are limited by severe side-effects after long-time administration. While nano-drug delivery systems present as promising strategies for INH delivery, the therapeutic efficacies are usually suboptimal due to ineffective drug accumulation at diseased sites. Local delivery system can achieve high drug concentration at focus sites with minimal systemic exposure, and herein we aimed to employ this strategy to develop a novel liposome-in-hydrogel system for localized treatment of bone TB. To achieve sustainable drug release, a derivative of INH called DINH was loaded because of its hydrophobicity, as well as its better activity and higher biosafety than INH. The hybrid system was demonstrated for thermo-responsive and self-healing properties via phase transition test and rheological studies, which were particularly useful for intra-articular administration. In vivo microdialysis studies revealed that the system can rapidly release drug into synovial fluid to reach effective inhibitory concentrations after localized injection, followed by a steady-state drug release. The optical image studies were performed to study its long-term behavior in vivo, which suggested a sustained drug release profile for several days. This work provides a promising drug delivery system for bone TB therapy.

Introduction

Tuberculosis (TB) is a highly contagious chronic bacterial infection caused by Mycobacterium tuberculosis (M.tb) (Fogel, 2015). Bone TB is one of the most common forms of extrapulmonary TB (∼10%) (Leonard and Michael, 2011), which is routinely treated by surgical intervention, accompanied by indispensable antitubercular drug therapy (Pigrau-Serrallach and Rodriguez-Pardo 2013). Due to the excellent activity and selectivity for M.tb, as well as its low price, isoniazid (INH) has been widely used as a first-line anti-tubercular drug (Brossier 2011). However, conventional INH treatment is generally limited by systemic side-effects, rapid drug clearance, and non-specific drug distribution (Barth and Egelund, 2016). As patients are recommended to treat with INH for at least 9 months (it is even longer for children) (Menzies et al., 2018), the cumulative adverse-effects would significantly decrease patient compliance after long-time administration.

Currently, various INH delivery strategies have been developed to improve therapeutic index, and most of them involve nano-devices (Li et al., 2011, Bhandari and Kaur, 2013, Kaur and Singh, 2014, Zhu et al., 2015). While these systems are advantageous to optimize drug dosage and lessen side effects, they are usually suffered from burst drug liberation and short drug release duration partially due to the hydrophilic nature of INH. For example, Li and coworkers reported a chitosan-based hydrogel, while the drug completely released outside the hydrogel within 4 h (Li et al., 2011). Kaur designed a solid nanoparticle with sustained drug release lasted only 24 h (Bhandari and Kaur 2013).

Recently, a series of hydrophobic INH derivatives were synthesized (Kumar et al., 2014). N′-Dodecanoylisonicotinohydrazide (DINH) is one such structure that displays strong hydrophobicity (log P = 4.84), remarkably improved activity and biosafety. We reason that DINH is a good alternative to INH for construction of sustained drug delivery systems. Liposomes are a type of nano-drug delivery system that have made real clinic impact, with various products commercially available or undergoing clinical trials (Feldman et al., 2011, Chang and Yeh, 2012). Liposomes can load hydrophobic drugs in their lipid bilayer for sustained drug release (Schwendener and Schott, 2010, Lee et al., 2015, Vahed et al., 2017). presenting an excellent carrier for DINH.

While systemic administration of liposomes has showed good therapeutic efficacy for various diseases (Bardania et al., 2017, Zylberberg and Matosevic, 2017), the treatment of the bone TB is much more difficult due to the poor blood supply in bone TB sites (Horsburgh et al., 2015). To this end, localized administration is preferred as it is able to attain high drug concentration at focus site without systemic exposure (Wolinsky et al., 2012). For example, Ye and coworkers reported a hierarchical scaffold loaded with INH and rifampin for local multi-drug treatment of TB, and showed that effective drug concentrations were maintained up to 12 weeks but extremely low in blood (Zhu et al., 2015). However, the treatment was realized via surgical implantation, which brought extra pain to patients. To solve this problem, thermo-responsive hydrogels have been developed, which can be conveniently injected into the target sites and rapidly change into gel phase at body temperature (Jeong et al., 1997, Yu and Ding, 2008, Almeida et al., 2014). In addition, when the hydrogels are equipped with self-healing properties, the service life at diseased sites can be enhanced via self-repair after damage, thus decreasing the administration frequency (White et al., 2002). Moreover, the incorporation of liposomes into hydrogels would further extend the drug release time, as the drugs would experience two transportation barriers of liposome and hydrogel network during release (Billard et al., 2015, Grijalvo et al., 2016, O'Neill et al., 2017).

Herein, we constructed a liposome-in-hydrogel system as carrier for localized treatment of bone TB. The PLGA-PEG-PLGA copolymer, an extensively studied thermo-responsive material (Alexander et al., 2013, Miyazaki et al., 2017), was employed as hydrogel matrix (Scheme 1). The DINH loaded liposomes were incorporated into PLGA-PEG-PLGA hydrogel by physical mixing. The resulting system was characterized for thermo-responsive sol-gel transition, mechanical and rheological properties. Interestingly, the self-healing properties of PLGA-PEG-PLGA based hydrogel was demonstrated for the first time, which is beneficial for localized bone TB treatment. The system showed prolonged drug release properties in vitro, and the in vivo sustained drug release was demonstrated through microdialysis and optical imaging techniques.

Section snippets

Materials

PLGA-PEG-PLGA was purchased from Polyscitech Company (USA). Isoniazid, dodecanoyl chloride, 4-dimethylaminopyridine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were from Sigma-Aldrich (saint Louis, MO, USA). Phospholipid (PC) was obtained from Lipoid Company (Germany). Sodium deoxycholate monohydrate (SDC) and sodium dodecyl sulfate (SDS) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Chlorin e6 (Ce6) was from Frontier Scientific, Inc. (Utah,

One-pot synthesis of DINH

DINH was synthesized according to a previous report with a slight modification (Inset of Fig. 1A) (Kumar et al., 2014). INH was activated by N,N‐dimethylaminopyridine (DMAP) at acylhydrazine group, which allowed subsequent reaction with dodecanoyl chloride to produce DINH with a yield ∼80%. The product was purified and collected by silica gel column. From Electrospray Ionization Mass (ESI-MS) analysis, the purified product showed a single m/z peak at 320.46 ([M + H]), which was consistent with

Conclusions

In summary, we demonstrated a thermo-responsive and self-healing liposome-in-hydrogel system for localized treatment of bone TB. The drug loading liposomes were facilely prepared and incorporated into PLGA-PEG-PLGA hydrogel via simple mixing. The entrapment of liposomes did not disturb the characteristics of the hydrogel, while the resulting hybrid system showed several interesting properties, such as temperature-dependent sol-gel transition and self-healing ability, making it a promising

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by Innovation-Driven Project of Central South University (No. 20170030010004), State Key Laboratory of Powder Metallurgy in Central South University, National Natural Science Foundation of China (No. 81573374), Key Laboratory Breeding Base of Hunan Oriented Fundamental and Applied Research in Innovative Pharmaceutics (No. 2016TP1029), and Hunan Engineering Research Center for Optimization of Drug Formulation and Early Clinical Evaluation (No. 2015TP2005).

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